Detection of antibacterial activity in excretory secretory product of adult trichuris suis

ABSTRACT

The present invention provides a heat-stable and protease-resistant antibacterial activity in excretory-secretory products (ESP) of  Trichuris suis . The antibacterial activity is not more than 10,000 MW; is resistant to boiling, trypsin, and pronase E; has a bactericidal mode of action; and is effective against Gram positive and Gram negative bacteria, including  Escherichia coli, Campylobacter jejuni, Campylobacter coli , and  Staphylococcus aureus . The antibacterial activity is useful in applications for killing or inhibiting the growth of microorganisms, in particular bacteria.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to Provisional Application No.60/246,203, which was filed Nov. 6, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was supported by National Institutes of Health Grant No.AI42348-03. The U.S. government has certain rights in this invention.

REFERENCE TO A “COMPUTER LISTING APPENDIX SUBMITTED ON A COMPACT DISC”

Not Applicable.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to heat-stable and protease-resistantantibacterial activity in excretory-secretory products (ESP) ofTrichuris suis. The antibacterial activity is not more than 10,000 MW;is resistant to boiling, trypsin, and pronase E; has a bactericidal modeof action; and is effective against Gram positive and Gram negativebacteria, including Escherichia coli, Campylobacter jejuni,Campylobacter coli, and Staphylococcus aureus. The antibacterialactivity is useful in applications for killing or inhibiting the growthof microorganisms, in particular bacteria.

(2) Description of Related Art

Compounds with antibacterial activity have been identified from a widearray of invertebrates, including parasitic nematodes (Wardlaw et al.,J. Appl. Bacteriol. 76: 36-41 (1994); Kato, Zoo. Sci. 12: 225-30(1995)). These factors constitute a primitive humoral defense system. Itis not surprising that metazoan parasites inhabiting thegastrointestinal tract (GI) produce antibacterial substances, since theyare in a microbe-rich environment containing potential pathogens. Forexample, a potent antibacterial activity was found in the body fluid ofAscaris suum, a nematode parasitizing the pig small intestine (Wardlawet al., J. Appl. Bacteriol. 76: 36-41 (1994); Kato, Zoo. Sci. 12: 225-30(1995)). The bactericidal activity was heat stable and less than 14,000MW in size. Subsequently, three humoral defense activities(antibacterial, bacteriolytic, and agglutinating) were detected in thebody fluid of A. suum (Kato, Zoo. Sci. 12: 225-30 (1995)).

The A. suum antibacterial factor (referred to as ASABF) has beenwell-characterized (Kato and Komatsu, J. Biol. Chem. 271: 30493-30498(1996)). ASABF is a heat-stable and trypsin-sensitive peptide of 71amino acids (Kato and Komatsu, J. Biol. Chem. 271: 30493-30498 (1996)).ASABF has structural and functional similarities to the defensins ofinsects/arthropods. Both are cysteine-rich, cationic peptides that aremore effective against Gram positive bacteria than Gram negativebacteria (Kato and Komatsu, J. Biol. Chem. 271: 30493-30498 (1996)).ASABF has significant sequence identity with the proteins deduced from acDNA sequence (yk150c7) and from a putative gene (T22H6.5) ofCaenorhabditis elegans, a free-living nematode (Kato and Komatsu, J.Biol. Chem. 271: 30493-30498 (1996)).

A gene family of saposin-like proteins has been identified in C.elegans, with one of them (T07C4.4) having antibacterial activity whenexpressed as a recombinant in E. coli (Banyai and Patthy, Biochim.Biophys. Acta 1429: 259-64 (1998)). The putative products of these C.elegans genes are similar to the amoebapores of Entamoeba histolyticaand a putative amoebapore-related protein of the liver fluke Fasciolahepatica in that they consist of a single saposin-like domain and asecretory signal peptide (Banyai and Patthy, Biochim. Biophys. Acta1429: 259-64 (1998)). Amoebapores of E. histolytica, an invasiveprotozoan pathogen, are pore-forming peptides with antibacterial ancytolytic activities, which function by formation of ion channels intarget cell membranes (Andra, et al., FEBS Letters 385: 96-100 (1996);Leippe et al., Molec. Microbiol. 14: 895-904 (1994)).

Antibacterial activity in invertebrates is quite common. Antibacterialpeptides have been described from the silkworm, Bombyx mori (Chowdhury,et al., Biochem. Biophys. Res. Comm. 214: 271-8 (1995); Hara andYamakawa, J. Biol. Chem. 270: 29923-7 (1995); Kim et al., Biochem.Biophys. Res. Comm. 246: 388-92 (1998)). A proline-rich antibacterialpeptide from the earthworm, Lumbricus rubellus, has been reported (Choet al., Biochim. Biophys. Acta 1408: 67-76 (1998)). Antibacterial agentshave also been detected in two other annelid species, Nereisdiversicolor (Salzet-Raveillon et al., Cell. Molec. Biol. 39: 105-14(1993)) and Eisenia foetida (Kauschke and Mohrig, Devel. Compar.Immunol. 11: 331-41 (1987)).

Most of the antimicrobial agents identified from invertebrates arepeptides that exhibit structural similarities. Insect defensins arecationic, cysteine-rich peptides forming intra-molecular disulfidebridges that appear in the hemolymph after bacterial challenge or injury(Cociancich et al., J. Biol. Chem. 268: 19239-45 (1993)). They havepotent antibacterial activity against Gram positive bacteria mediated bydisruption of the permeability barrier of the cytoplasmic membrane(Cociancich et al., J. Biol. Chem. 268: 19239-45 (1993)). The myticinsfound in the haemocytes of the mussel, Mytilus galloprovincialis, arecysteine-rich and exemplify antibacterial peptides originating asprecursors with signal sequences that require proteolytic events toactivate the mature peptide (Mitta et al., Eur. J. Biochem. 265: 71-8(1999)). Antibacterial peptides offer a means for treating or preventingbacterial infections that is not based upon an antibiotic mode ofactivity.

Antibiotics are commonly used to prevent bacterial infections. However,because many antibiotics have been used with relative abandon over thepast half century, many microorganisms have developed resistance to manyof the antibiotics. As a consequence, many antibiotics have beenrendered ineffective at preventing bacterial infections. Unless newantibiotics or alternative antibacterial treatments are developed, theworld will be staring at a public health crisis of immense proportions.Therefore, there is a dire need for novel antibacterial compositions forpreventing bacterial infections.

SUMMARY OF THE INVENTION

The present invention provides at least one heat-stable andprotease-resistant antibacterial activity in excretory-secretoryproducts (ESP) of Trichuris suis. The antibacterial activity is not morethan 10,000 MW, has a bactericidal mode of action, and is effectiveagainst Gram positive and Gram negative bacteria, including Escherichiacoli, Campylobacter jejuni, Campylobacter coli, and Staphylococcusaureus. The antibacterial activity is useful in applications for killingor inhibiting the growth of microorganisms, in particular bacteria.

Therefore, the present invention provides an excretory-secretory productof Trichuris suis comprising at least one antibacterial activity whichinhibits the growth of Campylobacter jejuni as an assay strain in agrowth medium and wherein the antibacterial activity is resistant toboiling, freeze-thawing, trypsin, and pronase E.

The present invention further provides a method for producing anexcretory-secretory product with at least one antibacterial activitywhich inhibits the growth of Campylobacter jejuni as an assay strain ina growth medium and which is resistant to boiling, freeze-thawing, andproteases which comprises (a) culturing Trichuris suis in vitro in aserum-free medium; and (b) separating the excretory-secretory productwith the antibacterial activity from the medium.

Further still, the present invention provides a method for inhibiting amicroorganism which comprises contacting the microorganism with anexcretory-secretory product of Trichuris suis which comprises at leastone antibacterial activity which inhibits the growth of Campylobacterjejuni as an assay strain in a growth medium and which is resistant toboiling, freeze-thawing, trypsin, and pronase E in an amount whichinhibits the microorganism. In a particular embodiment of the method,the microorganism is a bacterium.

Further still, the present invention provides a composition comprisingat least one antibacterial activity isolated from excretory-secretoryproducts of Trichuris suis which inhibits the growth of Campylobacterjejuni as an assay strain in a growth medium and wherein theantibacterial activity is resistant to boiling, freeze-thawing, trypsin,and pronase E in a carrier. Preferably, the carrier is selected from thegroup consisting of filler, non-toxic buffer, physiological salinesolution, water, alcohol, ointment, cream, gel, balm, paste, liquor,tincture, elixir, tablet, lotion, paste, capsule, spirit, and perenteralsolution.

The present invention further provides a method for isolating anisolated antibacterial activity from excretory-secretory products ofTrichuris suis, wherein the antibacterial activity inhibits the growthof Campylobacter jejuni as an assay strain in a growth medium andwherein the antibacterial activity is resistant to boiling,freeze-thawing, trypsin, and pronase E, comprising (a) culturing theTrichuris suis in vitro in a serum-free medium to produce theexcretory-secretory products; (b) removing the antibacterial activityfrom components of the secretory-excretory products which are greaterthan 10,000 MW; (c) separating the antibacterial activity removed fromthe secretory products which are greater than 10,000 MW by columnchromatography; and (d) eluting the antibacterial activity from thecolumn to produce the isolated antibacterial activity wherein theantibacterial activity inhibits the growth of Campylobacter jejuni as anassay strain in a growth medium and wherein the antibacterial activityis resistant to boiling, freeze-thawing, trypsin, and pronase E.Preferably, the column chromatography is HPLC and wherein the HPLC usesa reverse phase C18 column and a gradient to separate the antibacterialactivity, preferably, wherein the gradient is a 5-80% acetonitrilegradient in 0.1% trifluoroacetic acid. Preferably, the antibacterialactivity is eluted from the HPLC column at between about 1 to 10minutes, at between about 40 to 50 minutes, or at between about 60 to 70minutes.

In any one of the above embodiments of the present invention, theantibacterial activity has a minimum inhibitory concentration and aminimum bactericide concentration of the bacterial activity which areabout the same.

Further still in any one of the above embodiments of the presentinvention, the excretory-secretory product of the Trichuris suis has anantibacterial activity which is not more than 10K MW.

Further still in any one of the above embodiments, the antibacterialactivity inhibits at least 50% of at least one species of cream positiveor cream negative bacteria. In particular, the above embodiment whereinthe antibacterial activity inhibits a bacteria selected from the groupconsisting of Campylobacter jejuni, Campylobacter coli, Escherichiacoli, and Staphylococcus aureus.

OBJECTS

It is an object of the present invention to provide an antibacterialactivity from the excretory-secretory products of Trichuris suis for usein antibacterial applications.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the inhibitory effect of ESP on the growth ofCampylobacter jejuni was proportional to the ESP concentration in anagar diffusion assay. The disks were saturated with ESP at 4, 3, 2, and1 mg ESP protein/ml corresponding to 80, 60, 40, and 20 μg total ESPprotein/disk (top row), cRPMI containing BSA at concentration equivalentto the ESP (middle row), and cRPMI without dilution (bottom disk). Therewas no growth inhibition with cRPMI or cRPMI containing BSA.

FIG. 2A shows that fractionation of the original Trichuris suis ESP on aC18 reverse phase HPLC column with a 5-80% ACN gradient in 0.1% TFAresulted in at least three fractions with antibacterial activity(identified by asterisks). The ESP sample was 100 μg in 0.02 ml andantibacterial activity was determined by the qualitative brothmicrodilution assay.

FIG. 2B shows that fractionation of the CAP standard on a C18 reversephase HPLC column with a 5-80% ACN gradient in 0.1% TFA resulted in onefraction with antibacterial activity (identified by the asterisk). TheCAP standard was 0.2 μg in 0.02 ml and antibacterial activity wasdetermined by the qualitative broth microdilution assay.

FIG. 3A shows MS of the ESP 20 minute fraction. The mass spectra wasobtained by bombarding samples in a glycerol matrix with a beam of Xeatoms. The boxes indicate the characteristic patterns for CAP.

FIG. 3B shows MS of the CAP standard 20 minute fraction. The massspectra was obtained by bombarding samples in a glycerol matrix with abeam of Xe atoms. The boxes indicate the characteristic patterns forCAP.

FIG. 4 shows the theoretical calculations estimating the residual CAP inthe original ESP. Log PC values were used to predict the maximum amountof CAP absorbed by the aggregate whipworm volume (4% of the totalsystem) and then released back into the medium using the worst-casescenario, i.e., that 100% was released.

FIG. 5A shows that the CAP-free ESP 10K retentate fractionated on areverse-phase C18 HPLC column with a 5-80% ACN gradient in 0.01% TFA hadno antibacterial activity. Sample was 10 μg in 0.02 ml and antibacterialactivity was determined by the qualitative broth microdilution assay.

FIG. 5B shows that the CAP-free ESP 10K filtrate fractionated on areverse-phase C18 HPLC column with a 5-80% ACN gradient in 0.01% TFA hadtwo fractions with antibacterial activity. Sample was 10 μg in 0.02 mland antibacterial activity was determined by the qualitative brothmicrodilution assay.

FIG. 6A shows that the HPLC profile for the original ESP 10K retentatefractionated on a reverse-phase C18 HPLC column with a 5-80% ACNgradient in 0.01% TFA. Sample was 10 μg in 0.02 ml.

FIG. 6B shows that the HPLC profile for the CAP-free ESP 10K retentatefractionated on a reverse-phase C18 HPLC column with a 5-80% ACNgradient in 0.01% TFA. Sample was 10 μg in 0.02 ml.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The initial observation that the excretory-secretory products (ESP) fromTrichuris suis, a parasitic nematode found in the large intestine ofpigs, contained novel antibacterial activity was in a tissue cultureinvasion assay against Campylobacter jejuni. Herein, is disclosed thenovel antibacterial activity of the present invention isolatable fromthe ESP collected from Trichuris suis adults cultured in vitro.

The term “trichuricin” refers to the novel antibacterial activityisolatable from the ESP of Trichuria suis. As shown herein, trichuricincomprises at least three novel antibacterial activities as determined byHPLC of a 10K filtrate of the ESP on a reverse phase C18 columndeveloped with a 5-80% ACN gradient in 0.1% TFA. The novel antibacterialactivities may be related or distinct. As used herein, trichuricinrefers to any one of the novel antibacterial activities and to anycombination of the three novel antibacterial activities.

The term “antimicrobial activity” refers to the ability of thetrichuricin of the present invention to inhibit or kill at least onespecies selected from the group consisting of Gram positive bacteria,Gram negative bacteria, fungi, and protozoans. In general, it isincreasingly preferred that the trichuricin inhibits or kills at least50%, 60%, 70%, 80%, 90% or all cells of at least one species of Grampositive or Gram negative bacteria, fungi, or protozoans. Sensitivebacteria include, but are not limited to, Campylobacter jejuni,Campylobacter coli, Escherichia coli, and Staphylococcus aureus.

The trichuricin of the present invention is separable from ESP by HPLCinto at least three fractions, each with antibacterial activity asdetermined by broth microdilution assays using Campylobacter jejuni33292 as the assay strain. The antibacterial activity of the trichuricincomprises at least three peptides, each of which has antibacterialactivity, and each of which are heat stable and resistant to digestionwith pronase E and trypsin. The trichuricin peptides each have amolecular weight of not more than about 10,000 Daltons as determined byfiltering the ESP through a filter with a 10K MW cut-off andfractionating the filtrate by HPLC. Trichuricin has the HPLC profileshown in FIG. 5B wherein a 0.2 ml volume of the 10K filtrate of the ESPis loaded onto a reverse phase C18 column and developed with a 5-80% ACNgradient in 0.1% TFA. As shown in FIG. 5B, the trichuricin antibacterialactivities are eluted from the HPLC column at between about 1 to 10minutes for antibacterial activity 1, between about 40 to 50 minutes forantibacterial activity 2, and between about 60 to 70 minutes forantibacterial activity 3 as shown in FIG. 5B. Trichuricin is expected tohave a bactericidal mode of action because the minimum inhibitoryconcentration (MIC) and the minimum bactericidal concentration (MBC) ofits antibacterial activity are about the same.

The specific activity of trichuricin varies from preparation topreparation because of the inherent variability attendant in preparingESP starting with infected pigs. However, in general, the unpurified ESPhas about 2,560 activity units (AU)/ml or 853 AU/mg protein (asdetermined by the inhibitory effect of the ESP on the Campylobacterjejuni 33292 test strain in broth microdilution assays as shown inTables 1 and 7). Further, as shown in Table 7, the 10K filter purifiedtrichuricin exhibited about 10,240 AU/ml or about 102,400 AU/mg protein(using Campylobacter jejuni 33292 as the test strain). The AU/ml is thereciprocal of the greatest serial dilution of ESP or filtrate thatinhibits the Campylobacter jejuni 33292. For example, a 50 μl aliquot ofthe 10K filtrate was serial diluted and the antibacterial activity ofeach dilution was determined. Because the 10K filtrate had 10,240 AU/ml,a 50 μl aliquot of the 10K filtrate for serial dilution contained 512 AUwhich was the reciprocal of the greatest serial dilution that still hadantibacterial activity. Therefore, the minimum antibacterialconcentration (MIC) of the antibacterial activity in the 10K filtrate is1 AU of a 10,240 AU/ml 10K filtrate preparation or about 10 ng ofprotein. In contrast, the MIC of the antibacterial activity in the ESPwas 1 AU of a 2,560 AU/ml ESP preparation or about 1.2 μg of protein. Inlight of the above, the minimum antibacterial activity of thetrichuricin is 1 AU using Campylobacter jejuni 33292 as the standard.

The novel antibacterial activity of the trichuricin, which is at leastactive at a neutral pH and at 37° C., inhibits the growth of both Grampositive and Gram negative bacteria. In particular, the novelantibacterial activity inhibits the growth of Campylobacter jejuni,Campylobacter coli, Escherichia coli, and Staphylococcus aureus.

The trichuricin of the present invention enables the treatment orcontrol of microbial infections caused by those organisms which aresensitive to trichuricin. Such treatment or control includesadministering to a host or tissue susceptible to or afflicted with amicrobial infection an antimicrobially effective amount of thetrichuricin. Preferably, the trichuricin includes all threeantibacterial activities when used to treat or control microbialinfections.

A antimicrobially effective amount of the trichuricin can be readilydetermined according to methods known in the art. For agricultural use,the composition comprises an antimicrobially effective amount of thetrichuricin and an agriculturally acceptable carrier suitable for theorganism (e.g., plant) to be treated. For example, for use in apharmaceutical composition, the trichuricin can have an ED₅₀ in vitroless than about 10⁻³ M. One with ordinary skill in the art can readilydetermine an antimicrobially effective amount of the trichuricin againsta target bacterial strain, for example, based on the ED₅₀ using themethods disclosed herein and the teachings of the art.

Because of the antibacterial properties of the trichuricin, it can alsobe used as a preservative or a sterilant of materials susceptible tomicrobial contamination. For example, an antimicrobially effectiveamount the trichuricin of the present invention can be used as adisinfectant for treating surfaces and can be incorporated intocompositions such as foods, cosmetics, animal feeds, and the like toimpart an antibacterial activity to the composition which preventsspoilage of the composition.

Furthermore, pharmaceutical compositions comprising the trichuricin ofthe present invention as an active ingredient in an antimicrobiallyeffective amount to produce the antibacterial effect in a susceptiblehost or tissue and a pharmaceutically acceptable, non-toxic sterilecarrier can be readily prepared based on the teachings provided hereinand in the art. Such carriers can be fillers, non-toxic buffers,physiological saline solution, and the like, including, but not limitedto, water, alcohol, solvents and oils in the form of aromatic waters,liquors, solutions, ointment, cream, gel, balm, paste, tinctures,elixirs, spirits, and perenteral solutions. The preparation can be usedtopically or systemically and may be in any suitable form such asliquid, solid or semi-solid, which includes injectable solutions,tablets, ointments, lotions, pastes, capsules and the like. For example,the trichuricin can be incorporated into an ointment and used as atopical antibacterial application to the skin, a cut, a scratch, orwound to prevent or treat an infection. In addition, the trichuricin canalso be administered in combination with other adjuvants or compatibledrugs where such a combination is seen to be desirable or advantageousin controlling an infection caused by harmful microorganisms.

Compositions comprising the antimicrobially effective amount oftrichuricin can be given via a route of administration suited to theform of the composition. Such compositions are, for example, in the formof usual liquid preparations including solutions, suspensions, emulsionsand the like which can be given orally, as a dental rinse, gingivally,topically, intravenously, subcutaneously, or intramuscularly. Thecomposition is administered in an antibacterially effective amount,generally a dose of about 0.01 to about 100 mg/kg/day, calculated asprotein is expected to be useful. However, the optimum upper and lowertherapeutic amounts and any contra-indications have not yet been fullyestablished.

Antibiotics are commonly used to prevent bacterial infections. However,many microorganisms have developed resistance to many of theantibiotics. As a consequence, many antibiotics are no longer effectiveat preventing bacterial infections. While new antibiotics may bedeveloped which will prevent bacterial infections, it would be moredesirable to develop bactericides which in general are more difficultfor bacteria to develop resistance to. Because the trichuricin of thepresent invention is bactericidal in nature, trichuricin is animprovement over antibiotics as a means for preventing bacterialinfections.

To prepare trichuricin from pig intestines, pigs are infected with about2500 viable Trichuris suis eggs by oral gavage. After about 45 to 50days, the pigs are killed 45 to 50 days after infection. The entire GItract is removed from the pig and the GI tract slit open longitudinally,the contents emptied into a bucket, and adult whipworms pickedindividually from the colon with forceps into successive petri dishes ofsterile saline pre-warmed to 37° C. The intestinal contents are washedthrough a 2 mm sieve with tap water and additional whipworms arerecovered from the sieve. All damaged or immature whipworms areeliminated and any remaining debris adhering to the whipworms isremoved.

After washing the whipworms in sterile saline, the whipworms are washedin sterile media such as Hanks balanced salt solution (HBSS) to removefine debris not visible under the microscope. This is followed byincubation in a 5×-concentrated antibiotic cocktail containing 500 U/mlpenicillin (PEN), 500 μg/ml streptomycin (STREP) and 1.25 μg/mlamphotericin B (AMB) in a medium such as RPMI-1640 for a 16 to 24 hrperiod. A second incubation in a 1× antibiotic cocktail containing PEN,STREP, and AMB is performed for an additional 16 to 24 hr period. Thewhipworms are then washed repeatedly in sterile media, at least 3changes for a minimum of 2 hr each, to remove residual antibiotics.Finally, the whipworms are incubated for 10 days in a media such asRPMI-1640 containing 1% glucose (about 4 whipworms/ml) at 37° C. withhumidified 5% CO₂ for production of whipworm-conditioned mediacontaining ESP.

To confirm sterility of the whipworm-conditioned media containing ESP,aliquots are plated on blood agar plates and incubated aerobically andanaerobically at 37° C. for at 48 hrs or more. Contaminated batches arediscarded. Whipworm-conditioned media containing the ESP are collecteddaily, pooled, and filtered through a 10,000 MW cutoff filter to producea solution containing the trichuricin free of higher molecular weightmaterial. The filtrate is sterile filtered and stored at −80° C. untiluse.

The trichuricin is further purified from the filtrate by HPLC. Thefiltrate is loaded onto a reverse phase C18 column preferably packedwith a Vydak 300 angstrom resin and developed with a 5-80% acetonitrile(ACN) gradient in 0.1% trifluoroacetic acid (TFA). The trichuricinantibacterial activity is eluted from the HPLC column as a firstantibacterial activity at between about 1 to 10 minutes, as a secondantibacterial activity at between about 40 to 50 minutes, and as a thirdantibacterial activity at between about 60 to 70 minutes. The elutedtrichuricin fractions are pooled, sterile filtered, and stored at −80°C. until use. Alternatively, each trichuricin elution fraction isseparately sterile filtered and stored at −80° C. until use.

As is readily apparent, it would be desirable to be able to produce thetrichuricin without relying on infected pigs. Therefore, the trichuricinis preferably produced by a recombinant organism containing DNA encodingthe trichuricin. The recombinant organism includes, but is not limitedto, a yeast wherein the DNA encoding the trichuricin is integrated intothe yeast genome, a transgenic plant wherein the DNA encoding thetrichuricin is integrated into the plant genome, a mammalian cellwherein the DNA encoding the trichuricin is in an expression vector suchas the Simliki forest virus expression vector, an insect cell whereinthe DNA encoding the trichuricin is in a baculovirus expression vector,and a bacterium which is not susceptible to the trichuricin or abacterium wherein the promoter for expressing the DNA encoding thetrichuricin is an inducible promoter such as the lacZ promoter.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

This example illustrates the discovery and subsequent isolation of thenovel trichuricin from the ESP of Trichuris suis and further provides apreliminary analysis of the novel trichuricin.

Materials and Methods

Experimental infection of pigs and recovery of adult Trichuris suis.Weaned pigs, were maintained in confinement housing and provided pigchow and water ad libitum. The pigs were experimentally infected withapproximately 2500 viable Trichuris suis eggs by oral gavage. Pigs werekilled 45 to 50 days after infection with a captive bolt gun. Thenecropsy procedure involved opening the abdomen and removing the entireGI tract. The GI tract was slit open longitudinally, contents emptiedinto a bucket, and adult whipworms plucked from the colon with forceps.Intestinal contents were washed through a 2 mm sieve with tap water andadditional whipworms were recovered from the sieve. Whipworms werepicked individually using forceps into successive petri dishes ofsterile saline pre-warmed to 37° C. When all damaged or immaturewhipworms were eliminated and whipworms appeared clean visually, theywere examined under a dissecting scope and any remaining debris adheringto the whipworms was removed.

Preparation of Trichuris suis ESP. The Trichuris suis ESP used in theoriginal experiments was prepared from adult whipworms pulled free fromthe colonic mucosa as described above and in Hill et al. (Exper. Para.77: 170-8 (1993)). After washing in sterile saline, the whipworms werewashed in sterile Hanks balanced salt solution (HBSS) to remove finedebris not visible under the microscope. This was followed by incubationin a 5×-concentrated antibiotic cocktail in RPMI-1640 for a 16 to 24 hrperiod. The original 5× cocktail contained 500 U/ml penicillin (PEN),500 μg/ml streptomycin (STREP), 1.25 μg/ml amphotericin B (AMB), and 350μg/ml chloramphenicol (CAP). A second incubation in a 1× antibioticcocktail without CAP was performed for an additional 16-24 hr period.The whipworms were then washed repeatedly in sterile HBSS, at least 3changes for a minimum of 2 hr each, to remove residual antibiotics.Finally, the whipworms were incubated for 10 days in RPMI-1640containing 1% glucose (4 whipworms/ml) at 37° C. with humidified 5% CO₂for collection of whipworm-conditioned media containing ESP. To confirmsterility, aliquots of ESP were plated on blood agar plates andincubated aerobically and anaerobically at 37° C. for at least 48 hrs.Contaminated batches were discarded. ESP was collected daily, pooled,and concentrated at 4° C. by ultrafiltration using an AMICON stir cell(Millipore, Bedford, Mass.) with a 10,000 MW cutoff to 1/20th of theoriginal volume. The total protein content of 20×-concentrated ESP wasdetermined using the Bradford assay, which ranged from 3-4 mgprotein/ml. Concentrated ESP was sterile filtered (0.22 μm; Millipore,Bedford, Mass.) and stored at −80° C. As a control for the volumereduction step, RPMI-1640 media containing 1% glucose without whipwormswas concentrated under the same conditions. Bovine serum albumin (BSA)added to concentrated RPMI (cRPMI) was also used as a control forprotein content.

Bacterial strains and media. The antibacterial activity in Trichurissuis ESP was originally observed using Campylobacter jejuni (ATCC33292), and this was the strain on which most experiments were performedfor this preliminary characterization. Several Campylobacter jejuni andCampylobacter coli isolates were used for MIC determinations (Table 1).TABLE 1 MICs of Trichuris suis ESP on Campylobacter spp. Campylobacterjejuni Campylobacter coli Strain MIC Strain MIC  33292^(a) 1:128 16793681:32 33560 1:128 17010887 1:64 43430 1:256 18439 1:64 43470 1:128 19351:64 19084571   1:64  1777708 1:64 15046764   1:512 17140  1:12819094451   1:256 43473 1:64 43433 1:64  43474 1:64 33291 1:128 434791:32 43429 1:128 43482 1:64 49349 1:256 43134 1:64Suspensions of Campylobacter jejuni (about 5 × 10⁵ cfu) were added toserial two-fold dilutions of 20x-concentrated ESP. The MIC was thehighest dilution with no growth after 48 hours incubation.^(a) Campylobacter jejuni strain 33292 was used in subsequentexperiments as the test organism for characterization of theantibacterial activity in Trichuris suis ESP.Additional quality control organisms routinely used for antimicrobialsusceptibility assays were tested, including Enterococcus faecalis (ATCC29212), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC 27853), and Streptococcus pneumonia(ATCC 49619). Campylobacter jejuni and Campylobacter coli were grown onMueller-Hinton agar supplemented with 5% sheep blood or inMueller-Hinton broth (MHB) in humidified 5% CO₂ to achieve amicro-aerophilic atmosphere. S. pneumoniae was grown aerobically onblood agar plates; all other strains were grown on tryptic soy agarplates under aerobic conditions. All organisms were incubated at 37° C.

Antibacterial activity assays. Agar diffusion and broth microdilutionmethods were used to characterize the antibacterial activity ofTrichuris suis ESP (NCCLS. Methods for Dilution AntimicrobialSusceptibility Tests For Bacteria That Grow Aerobically. Fourth Ed.(1997)). For the agar diffusion assay, susceptibility discs weresaturated with 20 μl ESP samples at various concentrations and appliedto plates inoculated confluently with the test organism. Plates wereobserved for zones of growth inhibition surrounding each disc afterincubation for 24 hr for all organisms except Campylobacter spp., forwhich results were recorded after 48 hr. For the susceptible controlorganisms listed previously, the effect of the inoculum concentrationwas assessed. For this experiment, suspensions of the organisms at 10⁸cfu/ml were serially diluted to achieve 10⁷, 10⁶, and 10⁵ cfu/mlsuspensions for inoculation of agar plates.

A broth microdilution method in 96 well plates was used to obtainqualitative and quantitative measures of antibacterial activity.Qualitatively, 50 μl samples of ESP were added to 50 μl suspensions ofCampylobacter jejuni (containing about 5×10⁵ cfu, as determined bystandard serial dilution and plating) in 96 well plates. Plates wereevaluated for growth as indicated by the presence of either turbidity orpellet of bacterial cells in the bottoms of U-shaped wells after 48 hrincubation.

To quantify activity and determine the minimum inhibitory concentration(MIC) of ESP against test organisms, serial two-fold dilutions of20×-concentrated ESP were prepared in 50 μl volumes of MHB (n=2). Eachwell was then inoculated with 50 μl of Campylobacter jejuni suspended inMHB at a starting inoculum of about 5×10⁵ cfu, prepared from anovernight (20-24 hr) plate culture of actively motile, early 10 phaseorganisms. The MIC was assessed as the highest dilution of ESP thatresulted in no visible turbidity after 48 hr of incubation. Theantibacterial titer was defined as the reciprocal of the MIC and wasexpressed in activity units (AU) per ml for selected experiments. Foreach well showing diminished or no turbidity, a 50 μl aliquot wassubcultured onto a Mueller-Hinton blood plate and incubated for 48 hr.The minimum bactericidal concentration (MBC) corresponded to the highestdilution that showed no growth upon subculturing.

Stability. Unfractionated ESP was subjected to various treatments andthen bioassayed for antibacterial activity using the broth microdilution assay to evaluate the stability the antibacterial activity.Physical treatments included boiling for 15 minutes and freezing-thawing(3 cycles at −70° C.). Digestions with trypsin and pronase E (Sigma, St.Louis, Mo.) were performed with 1 mg/ml final enzyme concentration on100 AU/ml ESP at 37° C. for 6 hr. In this experiment, BSA (1 mg/ml) wasdigested as a control and run on a standard 15% SDS-PAGE gel to confirmthat the enzymes were functional under these conditions. Prior to thebioassay, enzymes were inactivated by boiling for 15 minutes. Controlexperiments were carried out without ESP to confirm that theheat-inactivated trypsin and pronase E were not inhibitory to growth ofthe test bacteria.

Ultrafiltration. Ultrafiltration methods were used to nominally sizefractionate ESP for an approximate determination of the molecular weightof the antibacterial agent(s). AMICON stirred cells under pressurizednitrogen gas were used to separate ESP into fractions above (retentate)and below (filtrate) 30,000 MW with a Millipore YM30 membrane(Millipore) and fractions above (retentate) and below (filtrate) 10,000MW with a Millipore YM10 membrane. Prior to bioassay, the concentratedretentate fraction was returned to its original volume in RPMI-1640media to match the volume of the filtrate so that comparisons ofrelative activity could be made without distortions due to volumedifferences. Antibacterial activity was assayed in the retentate andfiltrate fractions using the agar diffusion method as described above.

High performance liquid chromatography. A Model 173 high performanceliquid chromatography (HPLC) system (Perkin Elmer Applied Biosystems,Inc., Foster City, Calif.) at the Michigan State UniversityMacromolecular Structure Facility was used to fractionate Trichuris suisESP for preliminary isolation of the antibacterial activity (Hearn,Meth. Enzymol. 104: 190-212 (1984)). Samples (150 μl) of ESP were mixedwith an equal volume of 0.1% trifluoroacetic acid (TFA) and centrifugedat full speed (11,000×g) at room temperature for 15 minutes. Two hundredmicroliters of the clarified supernatant containing either about 100 μgor 10 μg total ESP protein was injected onto a C18 reverse phase column(0.8 mm in diameter and 250 mm long) packed with Vydak 300 Å resin (LCPackings, San Francisco, Calif.). Compounds were eluted by a continuouslinear gradient of 5% acetonitrile (ACN) in 0.1% TFA to 80% ACN in 0.1%TFA over a three-hour period at a flow rate of 0.04 ml/min. Absorbanceof the eluate was monitored at 214 nm, and fractions were collectedmanually as peaks were detected. Eluted fractions were dried in a SAVANTSPEEDVAC concentrator (Savant Instruments, Inc., Hullwood, N.Y.),reconstituted in 50 μl sterile ultrapure water, and filter sterilized(COSTAR SPIN-X columns, Corning Life Sciences, Corning, N.Y.) prior tobioassay for antibacterial activity by the broth microdilution assay.

To address the concern that one or more of the antibiotics used duringthe whipworm culture procedure could persist if to the final ESPpreparation, control HPLC experiments with antibiotic standards wereperformed for comparison to ESP chromatograms. Initially, 1×concentrations of PEN (100 U/μl), STREP (100 μg/ml), AMB (0.25 μg/ml),and CAP (70 μg/ml) were performed for screening purposes. A subsequentexperiment with CAP at 2 μg/ml was then carried out.

Mass spectrometry. Fast atom bombardment-mass spectrometry (MS) was usedas an assay for residual antibiotics in ESP potentially carried over inthe solution or adherent to the whipworms during preparation (Burlingameet al., Anal. Chem. 70: 647R-716R (1998)). Mass spectra were obtainedusing a JEOL HX-110 double-focusing mass spectrometer (JEOL USA,Peabody, Mass.) operating in the positive ion mode. Ions were producedby bombardment of samples in a glycerol matrix with a beam of Xe atoms(6 keV) or Cs⁺ ions (12 keV). The accelerating voltage was 10 kV and theresolution was set at 1000. The instrument was scanned in 30 secondsfrom m/z 50 to 1000.

Consideration of CAP contamination. After detection of residual CAP inthe original ESP, multiple measures were taken to exclude thepossibility that the antibacterial activity of the ESP was an artifactof CAP contamination. Using the worst-case scenario, the predictedmaximum amount of CAP absorbed and how much would be released into themedium if the whipworms dissolved (i.e., released 100%) was estimated.However, it was more likely that very little of the drug was releasedback into the medium due to the equilibrium (plateau) established duringuptake. For estimating the predicted amount, the log PC value(n-octanol/water partition coefficient) was used, which is a measure oflipophilicity, and anthelminthic drug absorption kinetic data in modelnematodes, Ascaris suum and Haemonchus contortus (Ho et al., Mol.Biochem. Para. 41: 153-65 (1990); Ho et al., Mol. Biochem. Para. 52:1-13 (1992); Ho et al., J. Pharma. Sci. 83: 1052-9 (1994)). It wasassumed that the aggregate whipworm volume of these eccentrically shapednematodes occupied 4% of the incubation well volume (4 whipworms/ml atabout 10 μl/whipworm).

PEN and STREP were also included in this assessment, although AMB wasexcluded because it is an antifungal agent, which does not inhibit thegrowth of Campylobacter jejuni or the other test bacteria used in thesestudies. The size of the drug was also taken into consideration.Additionally, an experiment to assess the heat stability of theantibiotics was performed.

The definitive test was to prepare ESP in the complete absence of CAPfor subsequent control experiments that would parallel selectedexperiments performed with the original ESP. For these experiments, alocal farm with naturally Trichuris suis-infected pigs was identified asa source of whipworms to prepare the CAP-free ESP. The same protocol wasused except that CAP was omitted from the 5× antibiotic cocktailtreatment. Also, the 10K filtrate was saved for experimentation.

Results

Discovery of antibacterial activity in the ESP. The original ESP fromTrichuris suis had a dose-dependent growth inhibitory effect onCampylobacter jejuni in the agar diffusion assay (FIG. 1). ESPconcentrations of 80, 60, 40, and 20 μg total protein/disk had growthinhibition zones of 27, 25, 18, and 13 mm, respectively. Control cRPMIand cRPMI containing BSA at concentrations equivalent to ESP did notinhibit the growth of Campylobacter jejuni. The MICs of theantibacterial activity from the ESP on several Campylobacter jejuni andCampylobacter coli isolates were determined (Table 1). Campylobacterjejuni strains were consistently 2- to 4-fold more sensitive to ESP thanCampylobacter coli isolates (Table 1). The MBC of ESP on Campylobacterjejuni 332Sf2 was equivalent to the MIC at 1:128.

To confirm that the antibacterial activity was not limited toCampylobacter spp., additional organisms were tested by the agardiffusion method (Table 2). TABLE 2 Preliminary Inhibitory Spectrum ofTrichuris suis ESP Sensitivity^(a) Test organism 20 μg^(b) 2 μg^(b)Escherichia coli (Gram pos.) + − Pseudomonas aeruginosa (Gram neg.) − −Staphylococcus aureus (Gram pos.) + − Streptococcus pneumoniae (Grampos.) − − Enterococcus faecalis (Gram pos.) − −Agar plates were inoculated from a suspension of test organismscontaining 10⁸ cfu/ml.^(a)Susceptibility is refereed to by classifying organisms as resistant(−) or sensitive (+) to ESP as determined by the agar diffusion assay.^(b)Susceptibility discs were saturated with 20 μl of ESP atconcentrations of 1 mg/protein/ml (20 μg total protein/disc) and 0.1 mgprotein/ml (2 μg total protein/disc).As shown in Table 2, two of the five quality control test organisms, oneGram-negative and one Gram-positive, were sensitive to ESP. Escherichiacoli had a zone of growth inhibition 9 mm in diameter, whereasStaphylococcus aureus had a zone of 11 mm. Both organisms were sensitiveonly to the highest concentration of ESP tested (20 μg totalprotein/disk).

In a subsequent experiment, inocula of the susceptible quality controlorganisms were diluted to determine if sensitivity to ESP increased withdecreasing numbers of organisms present. As shown in Table 3, the zonesof growth inhibition were the same for all three inoculum sizes. TABLE 3Effect of Inoculum Size on ESP Sensitivity Inoculum Test organism1:10^(a) 1:100 1:1000 Escherichia coli   ⁹ ^(b) 9 9 Staphylococcusaureus 11 11 11^(a)A suspension of organisms at 10⁸ cfu/ml was serially diluted toachieve 10⁷, 10⁶, and 10⁵ cfu/ml suspensions for inoculation of agarplates.^(b)Diameters of growth inhibition zones were measured in mm.

Stability of the antibacterial activity in the ESP and ultrafiltrationof the antibacterial activity. As shown in Table 4, the antibacterialactivity of the original Trichuris suis ESP was unaltered by heattreatment and repeated freeze-thawing. Furthermore, Table 4 shows thatdigesting the ESP with trypsin and pronase E did not abolishantibacterial activity. When the ESP was filtered through a 30K filter,the fractions above and below 30,000 MW contained antibacterial activity(Table 4). However, the antibacterial activity was lost in the 30Kretentate after repeated dialysis. Sub-fractionation of the 30K filtrateon a 10,000 MW membrane also showed antibacterial activity in theretentate and filtrate. TABLE 4 Physical and Chemical Properties of ESPantibacterial Activity Treatment Activity^(a) Boiling + Freeze-thawing +Trypsin + Pronase E + 30K retentate + 30K filtrate + 10K retentate + 10Kfiltrate + 30K retentate dialyzed^(b) −^(a)Antibacterial activity was measured qualitatively by the brothmicrodilution assay using Campylobacter jejuni as the test organism.Activity was classified as no growth (+) or growth (−).^(b)The 30K retentate was dialyzed and concentrated 5x in 100x volumesof RPMI-1640 on a YM30 membrane.

HPLC fractionation of the antibacterial activity in the ESP and massspectrometry analysis of the antibacterial activity. Fractionation ofthe original ESP on a C18 reverse phase HPLC column showed that theantibacterial activity resided in several fractions with retention timesranging from about 20 minutes to over 100 minutes (FIG. 2A). ControlHPLC experiments to test for residual antibiotics revealed that CAPco-eluted with the 20 minute ESP fraction (FIG. 2B). The other twoantibiotics (PEN and STREP) and the antifungal agent (AMB) had no peaksmatching those present in ESP (data not shown). By comparison to a CAPstandard, mass spectrometry confirmed that the 20 minute ESP fractioncontained CAP or a CAP-like molecule (FIGS. 3A and 3B). Comparison ofpeak heights in the ESP and CAP standard for HPLC and mass spectrometryexperiments indicated that ESP contained less than 2 μg/ml residual CAPor a CAP-like molecule.

Consideration of CAP contamination in the ESP and removal of the CAPcontamination from the ESP. The log PC of PEN indicated that it was toohydrophobic to be released back into the media following absorption bythe whipworms (Table 5). In addition, PEN was inactivated by heattreatment (Table 6), which indicated that it was not be responsible forthe heat-resistant activity in ESP. Although boiling did not inactivateSTREP (Table 6), the log PC indicated that it was too hydrophilic to beabsorbed by the nematode in any significant quantity (Table 5). Thelarger size of STREP would also contribute to its relative exclusionfrom absorption across the cuticle (Table 5). TABLE 5 PhysicalProperties of Antibiotics Antibiotic MW log PC Streptomycin sulfate728.70 −9.53(—) Chloramphenicol 323.13  0.92(8.3x) Penicillin G 334.4  185(71x)Molecular weight (MW) and n-octanol/water partition coefficient (log PC)were compiled from a drug database at Upjohn. Concentrating factors arein parentheses.

TABLE 6 Effect of Boiling On Antibiotics Antibiotic Activity^(a)Streptomycin sulfate + Chloramphenicol + Penicillin G −Antibiotics (1x concentration) were boiled for 10 minutes.^(a)Antibacterial activity was measured qualitatively by the brothmicrodilution assay using Campylobacter jejuni as the test organism.

However, the log PC of CAP predicted that it could be absorbed and thenreleased by the whipworms, and further, CAP is not heat-inactivated(Tables 5 and 6). Therefore, an estimate of the maximum concentration ofCAP in the trace levels of CAP remaining in the ESP after every step inthe ESP production protocol was calculated (FIG. 4). The maximumconcentration of CAP in the final ESP preparation after three washeswith HBSS was estimated to be between about 1 to 2 μg CAP/ml. Theestimate is consistent with the HPLC and mass spectrometry data. Also,it was experimentally determined that the MIC of CAP for Campylobacterjejuni was about 2 μg/ml. However, because the antibacterial activitywas detectable in ESP diluted 1:100, the trace levels of CAP present inthe final ESP was insufficient to account for the antibacterial activityin the ESP. A 1:100 dilution of ESP reduces the concentration ofresidual CAP to approximately 0.01 to 0.02 μg/ml, which is about 100×less than its MIC.

Although the protein yield of the CAP-free ESP was low, which wasattributed to the limited nematode recovery from naturally infectedpigs, antibacterial activity was clearly present. Despite thediscrepancy in protein concentration, the antibacterial titers of thegreater than 10,000 MW fraction of original ESP and CAP-free ESP wereidentical at 2560 AU/ml (Table 7). However, of particular interest wasthe observation that a high level of antibacterial activity (10,240AU/ml) was in the 10K filtrate of the CAP-free ESP (Table 7). Comparisonof the HPLC profiles from the 10K retentate and filtrate of the CAP-freeESP showed that the majority of the material was in the 10K filtrate,including the antibacterial activity (FIGS. 5A and 5B). TABLE 7Comparison of Antibacterial Titers in Original and CAP-Free ESP[protein]^(a) 10K retentate^(b) 10K filtrate^(b) ESP 3 2560   320^(c)CAP-free ESP 0.1 2560 10,240^(a)Concentration of protein is mg/ml^(b)Activity in units/ml was calculated from the reciprocal of the MIC.^(c)Secondary filtrate because the original was not available.

The antibacterial activity in CAP-free ESP had characteristics identicalto the original ESP in that it also was resistant to boiling,freezing-thawing, and inactivation by trypsin and pronase E (Table 8).Additionally, HPLC chromatographs of the 10K retentates from theoriginal and CAP-free ESP were similar, with the exception of themissing 20 minute CAP peak (FIGS. 6A and 6B). TABLE 8 Physical andChemical properties of CAP-Free ESP Antibacterial Activity TreatmentActivity^(a) Boiling + Freeze-thawing + Trypsin + Pronase E +^(a)Antibacterial activity was measured qualitatively by the brothmicrodilution assay using Campylobacter jejuni as the test organism.Activity was classified as no growth (+) or growth (−).

Discussion

As shown by this example, a potent antibacterial activity in ESP wasisolated from adult Trichuris suis, a nematode which parasitizes thelarge intestine of swine. However, because ESP from an axenic culture ofTrichuris suis cannot be prepared without initially high concentrationsof antibiotics to prevent the overgrowth of fecal organisms associatedexternally and internally with the whipworms, it was imperative toexclude the antibiotics as the source for the antibacterial activity inthe ESP. HPLC, mass spectrometry, the physicochemical properties of theantibiotics, and preparation of CAP-free ESP with antibacterial activityshowed that the antibacterial activity in the ESP was native toTrichuris suis and not a consequence of the antibiotics used to preparethe ESP.

After detecting residual CAP in the ESP by HPLC and mass spectrometry,the extent to which PEN, STREP, and CAP would be carried over into thefinal ESP was estimated. Uptake of anthelminthic drugs by GI nematodesoccurs primarily by absorption across the cuticle (Ho et al., Mol.Biochem. Para. 52: 1-13 (1992)). The cuticle is secreted by a singlelayer of cells that form the hypoclermis, and is composed of a densecollagen and collagen-like protein matrix containing negatively chargedaqueous-filled pores (Ho et al., Mol. Biochem. Para. 41: 153-65 (1990);Fetterer and Rhoads, Vet. Para. 46: 103-11 (1993)). The rate-determiningbarrier for drug absorption is at the interface of the hydrophilicporous cuticle matrix and the underlying lipophilic hypodermis (Ho etal., J. Pharma. Sci. 83: 1052-9 (1994)). Absorption of anthelminthiccompounds across the cuticle generally increases with lipophilicity ofthe agent and is also influenced by the: size of the drug. Moleculeswith M_(r) greater than 350 KDa are absorbed less efficiently thansmaller molecules (Ho et al., J. Pharma. Sci. 83: 1052-9 (1994)).

It is unlikely that residual PEN accounted for the antibacterialactivity in the ESP because of its log PC and its sensitivity toinactivation by boiling (ESP retained its antibacterial activity afterboiling). STREP was not a contaminant of the ESP because of its log PCand its size. The residual CAP that contaminated the original Trichurissuis ESP did not contribute significantly to the antibacterial activityin the ESP because the ESP antibacterial activity persisted in ESP inwhich the CAP had been diluted to about 100× less than its MIC, which isbelow the level at witch CAP antibacterial activity is measurable, andbecause the antibacterial activity was present in CAP-free ESP.

It was considered highly unlikely that antibiotics could persist intothe final ESP preparation, because the whipworms were washed extensivelyafter antibiotic treatments. Nevertheless, low levels of residual CAPwere detected by HPLC and confirmed by mass spectrometry. Thisobservation underscores the importance of being cautious about routinelyincluding antibiotics in the whipworm incubations. Therefore, it ispreferable that CAP not be added to media used to grow Trichuris suisfor preparation of ESP containing antibacterial activity.

The ESP antibacterial activity was effective against both Gram-negativeand Gram-positive bacteria. Sensitivity to the ESP antibacterialactivity was proportional to the concentration of ESP but was notinfluenced by the number of organisms in the inoculum. Because the MICand MBC of the ESP antibacterial activity against Campylobacter jejuniwere equal, the ESP antibacterial activity appears to be primarilybactericidal.

The presence of antibacterial activity in ESP which had been subjectedto the harsh conditions of boiling, freezing-thawing, and exposure totrypsin and pronase E, indicated that the antibacterial activity residedin one or more stable molecules. The HPLC data of the ESP and CAP-freeESP showed that the antibacterial activity comprises at least threecomponents, each with antibacterial activity. Although the antibacterialactivity was resistant to proteases, particularly the highly nonspecificpronase E, it is still possible that the antibacterial activity residesin one or more short peptides. Data from the ultrafiltration experimentsindicated that at least one of the components of the antibacterialactivity comprises molecules which are about 10,000 MW or less in size.

Genes encoding antibacterial peptides have been identified in a varietyof invertebrate species (Furukawa et al., Biochem. J. 15: 265-71 (1999);Park et al., Insect Biochem. Molec. Biol. 27: 711-20 (1997)).Alternatively, antibacterial activity is found in peptide fragmentsderived from larger proteins that have been degraded (Bellamy et al.,Biochim. Biophys. Acta 1121: 130-6 (1992); Strub et al., Eur. J.Biochem. 229: 356-68 (1995)). It has been shown that Trichuris suis ESPproteins are degraded by a constituent zinc metalloprotease, which alsoundergoes auto degradation (Hill et al., Exper. Para. 77: 170-8 (1993)).It is possible that the antibacterial activity of the ESP is adegradation product of a larger Trichuris suis protein.

For example, the mechanism of many antibacterial peptides ispore-formation in bacterial cell membranes (Lockey and Ourth, Eur. J.Biochem. 236: 263-71 (1996); Maget-Dana and Peypoux, Toxicol. 87: 151-74(1994); Leippe et al., Proc. Natl. Acad. Sci. USA 88: 7659-63 (1991);Leippe et al., Molec. Microbiol. 14: 895-904 (1994)). Therefore, apossible candidate for the source of the antibacterial activity of thetrichuricin would be degradation products of the presumed Trichuris suiscounterpart of the 50K pore-forming protein from Trichuris trichuria(TT50) (Drake et al., Proc. Royal Soc. Lon. Ser. B: Biol. Sci. 257:255-61 (1994); Drake et al., Proc. Royal Soc. Lon. Ser. B: Biol. Sci.265: 1559-65 (1998)). TT50 produces ion channels in lipid bi-layers andit contains multiple cysteine residues forming disulfide bonds, whichare a common feature of antibacterial peptides.

The trichuricin of the present invention is the first description of anovel antibacterial activity from Trichuris suis. Only one otherparasitic nematode, Ascaris suum, has been shown to possess anantibacterial activity. However, the antibacterial activity was from thebody fluid of Ascaris suum (Kato and Komatsu, J. Biol. Chem. 271:30493-30498 (1996)) and not the ESP. The possibility that theantibacterial activity was a component of the ESP, which had accumulatedin the body fluid of the Ascaris suum, is unlikely but cannot be ruledout.

EXAMPLE 2

This example shows the sequencing and synthesis of trichuricin.

The trichuricin is prepared and isolated from the ESP as in Example 1.The trichuricin is sequenced using peptide sequencing methods well knownin the art. The sequence of the trichuricin antibacterial activitiesenables degenerate PCR primers to be made which can be used to identifythe Trichuris suis genomic DNA encoding the antibacterial activities.The amino acid sequence allows synthetic trichuricin to be chemicallysynthesized using peptide synthesis technologies well known in the art.

EXAMPLE 3

This example shows the preparation of monoclonal antibodies thatrecognize the trichuricin.

The trichuricin is prepared and isolated from the ESP as in Example 1 orsynthesized as in Example 2. Then the trichuricin is used to makemonoclonal antibodies according to the methods in Antibodies, ALaboratory Manual. Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988), well known to those skilled inthe art as a source for methods for making polyclonal and monoclonalantibodies. Because of the small size of the trichuricin (less than orequal to 100 amino acids), the trichuricin is preferably coupled to acarrier such as bovine serum albumin or keyhole limpet hemocyanin with abifunctional reagent such as glutaraldehyde (amino to amino),m-Maleimidobenzoic acid-N-hydroxy-succinimide (MBS; amino tosulfhydryl), bisdiazo-benzidine (BDB; tyr to tyr), or carbodiimide(EDAC; amino to carboxyl) using methods well known in the art.

BALB/c mice are immunized with an initial injection of 1.0 μg of thetrichuricin per mouse mixed 1:1 with Freund's complete adjuvant. Aftertwo weeks, a booster injection of 1.0 μg of the trichuricin is injectedinto each mouse intravenously without adjuvant. Three days after thebooster injection the mouse serum is checked for antibodies to thetrichuricin. If positive, a fusion is performed with a mouse myelomacell line. Mid log phase myeloma cells are harvested on the day offusion, checked for viability, and separated from the culture medium bylow-speed centrifugation. Then the cells are resuspended in serum-freeDulbecco's Modified Eagle's medium (DMEM).

The spleens are removed from the immunized mice and washed three timeswith serum-free DMEM and placed in a sterile Petri dish containing 20 mlof DMEM containing 20% fetal bovine serum, 1 mM pyruvate, 100 unitspenicillin, and 100 units streptomycin. The cells are released byperfusion with a 23-gauge needle. Afterwards, the cells are pelleted bylow-speed centrifugation and the cell pellet is resuspended in 5 ml 0.17M ammonium chloride and placed on ice for several minutes. Then 5 ml of20% bovine fetal serum is added and the cells pelleted by low-speedcentrifugation. Afterwards, the cells are resuspended in 10 ml DMEM andmixed with myeloma cells to give a ratio of 3:1. The cell mixture ispelleted by low-speed centrifugation, the supernatant fraction removed,and the pellet allowed to stand for 5 minutes. Next, over a period of 1minute, 1 ml of 50% polyethylene glycol (PEG) in 0.01 M HEPES pH 8.1 at37° C. is added. After 1 minute incubation at 37° C., 1 ml of DMEM isadded for a period of another 1 minute, then a third addition of DMEM isadded for a further period of 1 minute. Finally, 10 ml of DMEM is addedover a period of 2 minutes. Afterwards, the cells are pelleted bylow-speed centrifugation and the pellet resuspended in DMEM containing20% fetal bovine serum, 0.016 mM thymidine, 0.1 hypoxanthine, 0.5 μMaminopterin, and 10% hybridoma cloning factor (HAT medium). The cellsare then plated into 96-well plates.

After 3, 5, and 7 days, half the medium in the plates is removed andreplaced with fresh HAT medium. After 11 days, the hybridoma cellsupernatant is screened by an ELISA assay. In this assay, 96-well platesare coated with the trichuricin. One hundred μl of supernatant from eachwell is added to a corresponding well on a screening plate and incubatedfor 1 hour at room temperature. After incubation, each well is washedthree times with water and 100 μl of a horseradish peroxide conjugate ofgoat anti-mouse IgG (H+ L), A, M (1:1,500 dilution) is added to eachwell and incubated for 1 hour at room temperature. Afterwards, the wellsare washed three times with water and the substrate OPD/hydrogenperoxide is added and the reaction is allowed to proceed for about 15minutes at room temperature. Then 100 μl of 1 M HCl is added to stop thereaction and the absorbance of the wells is measured at 490 nm. Culturesthat have an absorbance greater than the control wells are removed to 2cm² culture dishes, with the addition of normal mouse spleen cells inHAT medium. After a further three days, the cultures are re-screened asabove and those that are positive are cloned by limiting dilution. Thecells in each 2 cm² culture are counted and the cell concentrationadjusted to 1×10⁵ cells/ml. The cells are diluted in complete medium andnormal mouse spleen cells are added. The cells are plated in 96-wellplates for each dilution. After 10 days, the cells are screened forgrowth. The growth positive wells are screened for antibody production;those testing positive are expanded to 2 cm² cultures and provided withnormal mouse spleen cells. This cloning procedure is repeated untilstable antibody producing hybridomas are obtained. Then the identifiedstable hybridomas are progressively expanded to larger culture dishes toprovide stocks of the cells.

Production of ascites fluid is performed by injecting 0.5 ml of pristaneintraperitoneally into female mice to prime the mice for ascitesproduction. After 10 to 60 days, 4.5×10⁶ cells are injectedintraperitoneally into each mouse and ascites fluid is harvested between7 and 14 days later.

Hybridomas that successfully produce monoclonal antibodies againsttrichuricin are expanded as above, and used to make monoclonalantibodies for purifying the trichuricin from ESP and for identifyingDNA library clones which express the trichuricin.

EXAMPLE 4

This example shows the preparation of polyclonal antibodies thatrecognize the trichuricin.

The trichuricin is prepared and isolated from the ESP as in Example 1 orsynthesized as in Example 2. Then the trichuricin is used to makepolyclonal antibodies according to the methods in Antibodies, ALaboratory Manual. Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988), well known to those skilled inthe art as a source for methods for making polyclonal and monoclonalantibodies. Because of the small size of the trichuricin (less than orequal to 100 amino acids), the trichuricin is preferably coupled to acarrier such as bovine serum albumin or keyhole limpet hemocyanin with abifunctional reagent such as glutaraldehyde (amino to amino),m-Maleimidobenzoic acid-N-hydroxy-succinimide (MBS; amino tosulfhydryl), bisdiazo-benzidine (BDB; tyr to tyr), or carbodiimide(EDAC; amino to carboxyl) using methods well known in the art.

Rabbits are immunized preferably by the

Vaitukaitis protocol: multiple intradermal immunizations at once. Theupper back of the rabbit is shaved prior to immunization. About 1 mg ofpeptide is used per immunization (3-5 intradermal sites at eachimmunization), and the rabbits re-immunized at 2-6 week intervals. Forthe initial immunization, the immunogen (peptide-carrier conjugate) ismixed with an equal volume of complete Freund's adjuvant. For boosts,the immunogen is mixed with incomplete Freund's adjuvant. After theinitial immunization and 2-3 boosts, the rabbits are bled 3-4 weekslater, and the antibodies tested by immunoprecipitation of ¹²⁵I-labeledpeptide.

The polyclonal antibodies are used for purifying the trichuricin fromESP and for identifying DNA library clones which express thetrichuricin.

EXAMPLE 5

This example shows the preparation of a DNA library that expresses thetrichuricin. The methods for making and screening DNA expressionlibraries are well known to those skilled in the art and are describedin Molecular Cloning: A Laboratory Manual, Second Edition. Sambrook etal. (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989). The monoclonal antibodies made as in Example 3 orpolyclonal antibodies made as in Example 4 are used to screen thelibrary for clones that express the trichuricin.

Clones encoding the trichuricin are used to provide DNA encoding thetrichuricin. The DNA, when under the control of an inducible promoter,is used to express the trichuricin in an expression vector in E. coli.The expressed trichuricin is purified for use.

Alternatively, DNA encoding the trichuricin is obtained by purifying thetrichuricin by HPLC as described in Example 1 and sequencing thetrichuricin. Degenerate PCR primers are made which are then used in aPCR reaction containing Trichuris suis genomic DNA to PCR amplify a DNAproduct containing nucleotide sequences encoding the trichuricin. Theamplified PCR product is used as a probe to identify Trichuris suisgenomic DNA and cDNA containing the nucleotide sequences encoding thetrichuricin. The DNA encoding the trichuricin is isolated and clonedinto a plasmid with a promoter for expressing the trichuricin. Theplasmid encoding the trichuricin is transformed into a suitable organismfor expression of the trichuricin.

EXAMPLE 6

The antibacterial activity of the trichuricin of the present inventionis purified from the ESP of Trichuris suis by HPLC as shown inExample 1. The HPLC fractions which are identified (as shown in FIGS. 2and 5) as containing the antibacterial activity are purified and used toproduce compositions, mixtures, and solutions which contain thetrichuricin of the present invention. Alternatively, the trichuricin isproduced from an expression vector or chemically synthesized.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1-10. (canceled)
 11. A method for inhibiting a microorganism whichcomprises: contacting the microorganism with an excretory-secretoryproduct of Trichuris suis which comprises at least one antibacterialactivity which inhibits the growth of Campylobacter jejuni as an assaystrain in a growth medium and which is resistant to boiling,freeze-thawing, trypsin, and pronase E in an amount which inhibits themicroorganism.
 12. The method of claim 11 wherein the microorganism is abacterium.
 13. The method of claim 11 wherein the antibacterial activityhas a minimum inhibitory concentration and a minimum bactericideconcentration of the bacterial activity which are about the same. 14.The method of claim 11 wherein the antibacterial activity which is notmore than 10K MW.
 15. The method of claim 11 wherein the antibacterialactivity inhibits at least 50% of at least one species of cream positiveor cream negative bacteria.
 16. The method of claim 15 wherein theantibacterial activity inhibits a bacteria selected from the groupconsisting of Campylobacter jejuni, Campylobacter coli, Escherichiacoli, and Staphylococcus aureus. 17-29. (canceled)